Fig. 1.

Stromal interaction molecule 1 (STIM1) knockdown in human lung microvascular endothelial cells (HLMVECs) prevents thrombin-induced store-operated Ca²⁺ entry (SOCE), vascular endothelial protein tyrosine phosphatase (VE-PTP) phosphorylation, permeability increase, and disassembly of VE-cadherin at endothelial adherens junctions (AJs). A: HLMVECs transfected with 100 nM sc-siRNA or STIM1-siRNA. 48 h after transfection, cells were used to determine STIM1 protein expression by immunoblot (IB) (top) or used to measure thrombin-induced endoplasmic reticulum (ER)-store Ca²⁺ release and Ca²⁺ release-activated Ca²⁺ entry (bottom). B: nontransfected HLMVECs or HLMVECs transfected with sc-siRNA or STIM1-siRNA were challenged with thrombin (25 nM). Cells were used for immunoprecipitation with VE-PTP pAb and blotted with phosphotyrosine mAb. Quantified data from 3 experiments are presented as the means ± SE ratio of phosphorylated:total protein. *P < 0.05; transfected compared with nontransfected or sc-siRNA-treated cells. C: HLMVECs were transfected with sc-siRNA or STIM1-siRNA. At 24 h after transfection, cells were plated on gold electrodes (see materials and methods). After 24 h, cells were washed and incubated in 1% FBS containing medium for 2 h, and then cells were challenged with medium or thrombin (25 nM). Values from 3 experiments are presented as means ± SE. D: HLMVECs transfected with 100 nM sc-siRNA or STIM1-siRNA and then challenged with thrombin (25 nM) were stained with anti-VE-cadherin pAb. Experiment was repeated 3 times. The bar graph represents quantitative analysis of VE-cadherin staining at cell-cell junctions. Data shown are means ± SE of relative fluorescent intensity (RFI) compared with untreated cells. n = 8–10 cells per group; ***P < 0.001, transfected compared with nontransfected or sc-siRNA-treated cells.